The biologically active peptide, somatostatin, has been localized in several hypothalamic and extrahypothalamic brain regions where it may function as a classical neurotransmitter or as a modulator of neural activity. We have studied somatostatin binding sites in rat brain by incubation of tissue sections with 125-I-tyr-l-somatostatin, Ultrofilm autoradiography, computerized microdensitometry and comparison with 125-I standards. Highest concentrations of somatostatin binding sites (fmol/mg protein) were found in the claustrum, central nucleus of the amygdala, deep layers of cerebral cortex, lateral olfactory nuclei, hippocampus, medial and lateral septal nuclei, and the medial habenula. Regulation of somatostatin binding sites may be studied as one approach to examining the involvement of brain somatostatin pathways in various pysiological and behavioral states.